Aims: The intestinal microbiota plays an important role in host metabolism via production of dietary metabolites. Microbiota imbalances are linked to type 2 diabetes, but dietary modification of the microbiota may promote glycaemic control. This study investigated whether differences in gut microbiota between control and diabetic mice influence the production of polyphenolic metabolites from wheat wholegrain (WW). Second, the study assessed whether changes in metabolite profiles affect pancreatic beta cell function. Methods: Faecal samples (1% (v/v)) from control or high‐fat high‐fructose fed (HFHF) mice were fermented in vitro with 0.1% (w/v) WW for 0, 6 and 24 h. Polyphenolic profiles were determined by UHPLC‐MS/MS and microbiota composition by bacterial 16S rRNA sequencing and qPCR. MIN6 beta cell apoptosis was assessed by measurement of caspase activity and insulin release by radioimmunoassay. Results: Levels of WW polyphenols were decreased in fermentation samples from HFHF with time and HFHF showed an overall dysbiotic microbiome profile (p < 0.05 vs control at 0 h, n=3). WW fermentation led to major changes in microbiota profile in control (6 h) and HFHF (24 h) groups, with increased diversity in HFHF (p < 0.05, n=3). Twenty hours incubation with 1% supernatant from both groups did not alter MIN6 cell apoptosis in response to palmitate and cytokines (p > 0.05, n=3). Insulin secretion was inhibited at 0 h by control, but not HFHF supernatant, and this effect was partly reverted at 24 h (p < 0.05, n=5). Conclusion: Our results suggest that a dysbiotic HFHF microbiota profile affects the production of polyphenolic metabolites with potential implications for beta cell function.